Protocol

Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Cloning by Homologous Recombination and High-Throughput Transformation

This protocol was adapted from "Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array," contributed by Tony R. Hazbun and John P. Miller, Chapter 37, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

Although described here as a means of producing baits and preys, the generation of an array of ORFs fused to any moiety can be achieved using this protocol. This procedure capitalizes on the asexual/sexual reproductive cycle of yeast cells. Laboratory yeast strains can be maintained indefinitely by asexual reproduction as one of two haploid mating types, MATa or MAT. However, overlaying MATa and MAT cells allows them to fuse and form diploid cells. Hence, the strategy involves transforming MAT cells with the Gal4 BD (bait) fusion plasmids and transforming MATa cells with the Gal4 AD (prey) fusion plasmids. The transformants are then mixed on a plate, or in liquid, and diploids are selected by plating them onto media lacking the marker nutrients for both of the plasmids. In this way, the prey transformations need only be performed once, and each bait protein can then be tested against the array of prey-containing strains using only one transformation per bait. The protocol is easily adapted to small-scale transformation of single microcentrifuge tubes for bait transformations.